A quinone-dependent phosphatase was previously purified to homogeneity from Clostridium sticklandii and its amino acid composition determined. The only known substrate for this phosphatase was p-nitrophenylphosphate. The recent observation that 32P could be cleaved from (32P) phosphocasein by this enzyme suggests that its natural substrate is a bacterial phosphoprotein. Hence a role in cellular regulation or in a phosphate transfer process is indicated. An affinity chromatographic procedure for direct isolation of the phosphatase from crude bacterial extracts was developed by covalently attaching a quinone analog (4-amino-2-methyl-1-naphthol) to Sepharose 4B.